The invention concerns specific antibodies to cardiac muscle troponin T, their production and use in an immunological reagent for the determination of myocardial necrosis.
The myofibrils of the striated muscle consist of two protein filaments which act in combination, the thick filaments have myosin as their main component and the thin filaments contain actin, tropomyosin and troponins. Troponin, a regulatory structural protein, aggregates to a complex in the cells and consists of three different proteins:
Troponin C (MW 18000) which binds calcium ions
Troponin I (MW 24000) a sub-unit binding actin
Troponin T (MW 37000) which complexes with tropomyosin.
The comparable nomenclature has historical reasons since originally the complex as such was purified and was looked upon as a single protein and denoted troponin. Later it was proven that troponin really consists of three different proteins. This nomenclature was then retained because of the spacial relationship between the proteins on the thin filament of the contractile apparatus and because of their cooperation with respect to the regulation of muscle contraction. They are, however, three different proteins which are functionally related to the other proteins of the contractile apparatus such as myosin or actin but which have different amino acid sequences.
During long lasting severe ischaemia or muscle cell necrosis troponin I reaches the blood plasma and is thus a parameter for such diseases which can also be used for diagnosis and monitoring analogous to the known infarction enzymes CK, CK-MB, GOT and LDH.
It has been shown that the determination of CK and CK-MB is not absolutely specific for an infarction and is only increased in the serum 80 to 90 hours after the infarction. GOT and LDH are also not specific for cardiac muscle since increased amounts are also found in the blood in many other diseases.
The disadvantage of a determination of cardiac troponin I is that normally serum already contains concentrations of troponin I at a level of 10 ng/ml (cf. B. Cummins, J. Mol. Cell. Card. 19 (1987), 999-1010 and B. Cummins, Clin. Invest. 113 (1987) 1333-1344). It turns out that a biphasic serum concentration occurs in a transmural infarction on average, troponin I was increased from the 4th to the 168th hour after the onset of pain in 37 patients with acute transmural infarction. Similar results were obtained in an animal model. Accordingly it follows that for troponin I the 10th to 50th hour after the occurrence of an infarction is the time interval for the absolute diagnostic sensitivity. Thus, apart from the limited sensitivity, due to variable serum levels of troponin I the clinically important monitoring for 10 days and more after the occurrence of the infarction cannot be achieved by the determination of troponin I.
It was therefore the object of the present invention to eliminate these disadvantages and to provide a method for the determination with which monitoring is possible for at least 150 hours (duration of absolute diagnostic sensitivity) in myocardial infarctions and other injuries to the cardiac muscle.
This object is achieved by a method for the determination of myocardial necrosis according to the immunoassay principle which is characterized in that a serum or plasma sample is incubated with at least one antibody to troponin T and a binding partner B for troponin T or for the antibody, in which either the antibody to troponin T or the binding partner B is labelled with a determinable group, the immunological complex which thereby forms is isolated and the determinable group is determined in the isolated or in the remaining phase as a measure for troponin T from the sample.
The binding partner B has to bind to the antibody to troponin T or to troponin T. B can be for example a second antibody to troponin T or troponin T from humans or animals or an analogue thereof which is bound by the antibody.
The sample is preferably incubated with an antibody to troponin T and a conjugate of a further antibody to troponin T and a determinable group, the immunological complex formed is isolated by separation of the phases and the determinable group is determined in one of the phases.
It is furthermore preferred to incubate the sample with an antibody to troponin T and a conjugate of troponin T and a determinable group, to isolate the immunological complex formed by separation of the phases and to determine the determinable group in one of the phases.
Surprisingly, it turned out that a significantly higher sensitivity can be obtained by a troponin T immunoassay in the determination of myocardial necroses (such as e.g. by cardiac infarction, ischaemia or angina pectoris) than by the determination of other parameters such as CK, CK-MB, GOT, LDH or troponin I. As established by the inventors the reason for this is that in contrast to other proteins of the contractile apparatus no serum concentration can be measured for troponin T up to the detection limit of the test (0.25 ng/ml) in normal patients (who have not suffered myocardial necroses).
This is particularly surprising since, because of the functional relationship between the troponins, a similar serum concentration to that for troponin I would be expected for troponin T. Furthermore, the serum concentration curve of troponin T differs significantly, for example in a transmural infarction, from the curve for troponin I. In contrast to troponin I the curve of the time course is in three phases instead of two phases and troponin T is found to be increased on average for up to 300 hours after the onset of pain. The time interval for absolute diagnostic sensitivity lasts from the 6th to the 195th hour. The time interval for the absolute diagnostic sensitivity is thus nearly four times as long as that known for troponin I.
All common immunoassays are in principle suitable for the immunological method of determination according to the present invention such as radioimmunoassay, enzyme-immunoassay, fluorescence immunoassay etc. Furthermore, all variants of these procedures such as competitive immunoassay, IEMA procedure etc. are applicable. A sandwich test has proven to be particularly effective for the determination of troponin T. In this test procedure an immobilized antibody to troponin T and a conjugate of an antibody to troponin T and a determinable group is used. The different variants of this test method as well as details for carrying out these procedures are described at length in the literature. However, other immunological methods of determination are also possible using the antibodies according to the present invention such as those described e.g. in the German patent application DE-A 38 34 766 and/or DE-A 38 22 750.
Within the scope of the invention an antibody is understood as a complete antibody, chimeric antibody, bivalent antibody or fragments thereof. The troponin T antibodies used can be polyclonal or monoclonal. Monoclonal antibodies are preferably used.
In the sandwich test the sample solution is incubated with at least two antibodies to troponin T as a preferred embodiment. The first antibody thereby mediates the binding to the solid phase. For this, this antibody can either be bound directly or via a spacer to the solid phase or it can be present in a soluble form and only be immobilized after the immunological reaction has been carried out. The second antibody can either be labelled directly with a certain group or it can be bound to the determinable group by a functional bond. For this the antibody can be bound to one partner of a specific binding pair and the determinable group can be bound to the other partner of the specific binding pair. A complex which contains the second antibody as well as the determinable group then forms during the reaction. The binding of the first antibody to the carrier (immobilization) is carried out according to methods known to the expert by adsorptive, chemical binding or by functional binding via a specific binding pair. In this case one partner of the binding pair is immobilized while the other partner is chemically bound to the antibody. The antibody can then be immobilized before or during the immunological determination reaction via this binding pair. Examples of such binding pairs are biotin-streptavidin/avidin, hapten-antibody, antigen-antibody, sugar-lectin, hapten-binding protein.
The competitive test is a further preferred variant of the test. In this case an antibody to troponin T which is immobilized either before or during the determination and a conjugate of troponin T and a determinable group are used.
Materials can be used as carrier materials for the immobilization of the antibodies according to the present invention or for the immobilization of troponin T such as e.g. tubes, plastic cuvettes, microtitre plates, beads or plastic microcarriers, such as polystyrene, vinylpolymers, polypropylene, polycarbonate, polysaccharides, silicons, rubber or treated glass (cf. e.g. E. T. Maggio xe2x80x9cEnzyme Immunoassayxe2x80x9d CAC. Press, Florida (1980), in particular pages 175178, EP-A-063064, Bioengineering 16 (1974), 997-1003, C. J. Senderson and D. V. Wilson, Immunology 20 (1971), 1061-1065). In particular a carrier material, especially polystyrene, coated with avidin or streptavidin is used and is preferably prepared as described in EP-A-0269092. The binding partner B likewise contains either troponin T or an analogue thereof or a monoclonal antibody capable of specific binding to troponin T and is labelled. The usual agents for the respective determination method are suitable for the labelling. Thus, radioisotopes such as 57 Co are used for the labelling in a radioimmunoassay. All enzymes which are usually used for an enzyme-immunoassay are suitable, for example, peroxidase or 0-galactosidase. The common fluorescent groups are suitable as a label for a fluorescence immunoassay. Details of these different test methods and variants of the procedures are known to the expert. The binding of the label to troponin T or to the antibody can be carried out in an analogous manner to the binding to the solid phase i.e. covalently or via a specific binding pair.
The antibody or troponin T is covalently bound to one of the above-mentioned binding partners according to methods known to the expert, for example via carbodiimide and hydroxysuccinimide.
Peroxidase (POD) is preferably used as the enzyme label.
A further embodiment of the invention is a process for the production of polyclonal antibodies capable of specific binding to cardiac muscle troponin T, whose cross-reaction to human skeletal muscle troponin T is less than 5% and is less than 2% to troponin I and other myofibrillar proteins. The process consists in immunizing experimental animals, preferably sheep, with human cardiac muscle troponin T in combination with an adjuvant over several months (preferably 4-6 months) with at least four immunizations at intervals of four to six weeks, isolating the raw serum and purifying it immunosorptively.
A further embodiment of the invention is a process for the production of monoclonal antibodies capable of specification binding to cardiac muscle troponin T whose cross-reaction to human skeletal muscle troponin T is less than 5% and is less than 2% to troponin I and other myofibrillar proteins. The process is characterized in that experimental animals, preferably in-bred mice, are immunized intraperitoneally over several months with human cardiac muscle troponin T in combination with an adjuvant with at least four immunizations at intervals of four to six weeks, B lymphocytes are isolated and fused with a permanent myeloma cell line, the clones are isolated and the antibodies are isolated from them.
Aluminium hydroxide together with Bordatella pertussis or Freund""s adjuvant is preferably used as the adjuvant.
The primary cultures of hybrid cells obtained during the fusion according to the well-known procedure of Kxc3x6hler and Milstein (Nature 256 (1975), 495-497) are cloned in the usual way e.g. using commercial cell sorters or by xe2x80x9climiting dilutionxe2x80x9d. Those cultures are used further which react positively to isolated cardiac muscle troponin T and to cardiac muscle troponin T in the serum of patients and which react negatively to troponin T from skeletal muscle. Hybridoma cell lines are obtained in this way which produce the monoclonal antibodies according to the present invention. These cell lines can be cultured according to known methods and the monoclonal antibodies produced therefrom can be isolated.
Examples of hybridoma cell lines obtained in this was are clone 7.1 A 12.2-22 (ECACC 89060901) and clone 8.1 F 6.7-2 (ECACC 89030308). The cell lines are deposited under their respective quoted numbers at the European Collection of Animal Cell Cultures, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts. SP4, OJG, GB. on Jun. 9, 1989 and Mar. 3, 1989, respectively,
The polyclonal or monoclonal antibodies thus obtained are distinguished by their low cross-reactivity to human skeletal muscle troponin T which is less than 5% and preferably below 2% and by a cross-reactivity to troponin I and other myofibrillar proteins of less than 2%. The polyclonal and monoclonal antibodies according to the present invention are especially suitable for the specific determination of myocardial necrosis in a sample such as serum or plasma. The antibodies can be used as such for these methods of determination or as chimeric antibodies or as fragments thereof, for example, Fab fragments which have the corresponding immunological properties. The term antibody is therefore understood to indicate complete antibodies as well as fragments thereof.